Davies KA , Fitzgibbon C , Young SN , Garnish SE , Yeung W , Coursier D , Birkinshaw RW , Sandow JJ , Lehmann WIL , Liang LY , Lucet IS , Chalmers JD , Patrick WM , Kannan N , Petrie EJ , Czabotar PE , Murphy JM .

R01 GM114409 NIGMS NIH HHS

	Fig. 1. Few MLKL orthologues reconstitute necroptotic signaling in mouse and human cells.Genes encoding human, mouse, rat, horse, pig, chicken, tuatara, frog and stickleback MLKL were stably introduced into Mlkl−/− mouse dermal fibroblast (MDF) (a) and MLKL−/− human U937 (b) cells and expressed upon doxycycline treatment (induced). Cells were either untreated (UT) or treated with a necroptotic stimulus (TNF, Smc mimetic, IDN-6556; TSI) to examine the capacity of each orthologue to reconstitute necroptotic signaling. Cell death was measured by propidium iodide (PI) uptake by flow cytometry. Data shown are mean ± SEM of independent experiments on one U937 cell line (n = 3 for mouse, rat, horse and chicken MLKL; n = 4 for human and frog MLKL; n = 5 for pig MLKL) or two biological replicate MDF lines (n = 6, except for n = 8 for mMLKL-FLAG). * represents statistical significance of p < 0.05 using a paired, two-tailed t-test: a mouse-FLAG p = 0.000015, horse p = 0.0017, pig p = 0.0290; and b human p = 0.0104, pig p = 0.0003. Source data are provided in a Source Data file. An example of the flow cytometry gating strategy used throughout this study is shown in Supplementary Fig. 2. Expression of introduced genes was verified by western blot (Supplementary Fig. 3).
	Fig. 2. Rat and horse MLKL pseudokinase domain structures diverge from mouse MLKL.a The structure of the human MLKL pseudokinase domain (PDB, 4MWI39) shows a conventional active kinase-like conformation. The K230–E250 salt bridge (equivalent of the K72-E91 PKA interaction) between the β3 ATP-binding lysine and the Glu in the αC helix (shown in dark blue throughout) is shown in the zoomed inset. b The mouse MLKL pseudokinase domain (from PDB 4BTF18) shows a more open conformation owing to the activation loop adopting an unusual helical conformation (pale blue), which displaces the αC helix (dark blue). An unconventional interaction between the β3 K219 and the activation loop Q343 results (zoomed inset). c The rat MLKL pseudokinase domain adopts an active protein kinase-like conformation, resembling that of the human structure. The β3 K219 forms a conventional salt bridge with the αC helix E239 (zoomed inset), rather than a hydrogen bond with the activation loop Gln observed in the mouse structure. d The horse MLKL pseudokinase domain shows a similar active protein kinase-like conformation, with the K228:E248 salt bridge shown in the zoomed inset. The horse structure exhibits previously unobserved features, including burial of the activation loop in the pseudoactive site and an additional helix in the β3-αC loop (displayed in more detail in Fig. 4).
	Fig. 3. Horse MLKL β3-αC loop and C-lobe residues facilitate mouse RIPK3 engagement.a The horse MLKL pseudokinase domain structure superimposed on the reported mouse MLKL pseudokinase:mouse RIPK3 kinase domain structure (PDB 4M69 (ref. 40)). A site in each of the N- and C-lobes of horse MLKL pseudokinase domain were identified as possible interfaces between horse MLKL and mouse RIPK3 (b, c). d Wild type or alanine substitutions of putative interface residues in horse MLKL were expressed in Mlkl−/− MDF cells and their capacity to induce death in the absence of stimulation (untreated, UT) or upon addition of a necroptotic stimulus (TSI) following doxycycline induction of protein expression was quantified by PI uptake. e Wild type or F373A mouse MLKL were expressed in Mlkl−/− MDF cells and their capacity to induce death evaluated as in panel d. Data in d, e are shown are mean ± SEM of ≥3 independent experiments for two biological replicate MDF lines (n = 6 for wild-type horse MLKL; n = 7 for S233A, R241A; n = 8 for R235A, F373A; and n = 9 for S237A, Y384A and mouse MLKL wild type). * represents statistical significance of p < 0.05 using a paired, two-tailed t-test: d horse MLKL wild type p = 0.0017, R236A p = 0.0162, S237A p = 0.0249, R242A UT uninduced vs UT induced p = 0.0236, R242A TSI uninduced vs TSI induced p = 0.0041; e wild-type mouse MLKL p = 0.0002. Source data are provided in a Source Data file.
	Fig. 4. Horse MLKL activation loop phosphorylation induces conformational flexibility.a The activity of phosphomimetic mutations, S345D in rat MLKL, or T356E-S357E in horse MLKL, or alanine substitution (T356A-S357A) in horse MLKL, was tested in MLKL−/− U937 and/or Mlkl−/− MDF cells upon doxycycline induction of expression, in the presence (TNF, Smac mimetic, IDN-6556; TSI) and absence (untreated; UT) of necroptotic stimuli. These data are plotted alongside wild-type controls from Fig. 1. b, c The activation loop of the horse MLKL pseudokinase domain is buried in the pseudoactive site in a position occupied by ATP in conventional protein kinases, such as ERK (PDB 4GT3) (d). e Wild-type horse MLKL or alanine substitution mutants of activation loop and adjacent pseudoactive site residues were stably introduced into Mlkl−/− MDF cells and the capacity to kill cells in the presence (TSI) or absence (UT) of necroptotic stimuli in the presence (induced) or absence (uninduced) of doxycycline-induced exogene expression quantified by PI uptake using flow cytometry. Data in a and e are shown as mean ± SEM of ≥3 independent experiments for each of two biological replicate MDF lines (n = 6 for all in a, n = 7 for T208A and Q355A-T208A, n = 8 for Y282A and Q355A) or one U937 line (n = 3). * represents statistical significance of p < 0.05 using a paired, two-tailed t-test: a wild-type horse MLKL p = 0.0017, rat MLKL S345D in MDFs UT uninduced vs UT induced p = 0.0089, rat MLKL S345D in MDFs TSI uninduced vs TSI induced p = 0.0035, rat MLKL S345D in U937s UT uninduced vs UT induced p = 0.0108 and rat MLKL S345D in U937s TSI uninduced vs TSI induced p = 0.00114. For e T208A p = 0.0013, Q355A p = 0.0099 and Q355A-T208A p = 0.0056. f A comparison of molecular dynamics simulations on horse MLKL reveals increased activation loop flexibility in the phosphorylated MLKL model. The x-axis shows residue numbers and the y-axis shows root mean square fluctuation (RMSF) across the simulation. The phosphorylated residues, pT356 and pS357, are shown in red. g A series of snapshots of phosphorylated horse MLKL show the phosphorylated activation loop moving out of the pseudoactive site. Zoomed insets show hydrogen bonds at various stages of the transition.

Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed

Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed
Adrain, New lives for old: evolution of pseudoenzyme function illustrated by iRhoms. 2012, Pubmed
Bossen, Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human. 2006, Pubmed
Brennan, A Raf-induced allosteric transition of KSR stimulates phosphorylation of MEK. 2011, Pubmed
Bussi, Canonical sampling through velocity rescaling. 2007, Pubmed
Cai, Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. 2014, Pubmed
Chen, Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death. 2014, Pubmed
Chen, Diverse sequence determinants control human and mouse receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling. 2013, Pubmed
Cho, Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. 2009, Pubmed
Davies, The brace helices of MLKL mediate interdomain communication and oligomerisation to regulate cell death by necroptosis. 2018, Pubmed
Dondelinger, An evolutionary perspective on the necroptotic pathway. 2016, Pubmed
Dondelinger, MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. 2014, Pubmed
Emsley, Features and development of Coot. 2010, Pubmed
He, Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. 2009, Pubmed
Hildebrand, A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction. 2020, Pubmed
Hildebrand, Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death. 2014, Pubmed
Huang, RosettaRemodel: a generalized framework for flexible backbone protein design. 2011, Pubmed
Huang, CHARMM36 all-atom additive protein force field: validation based on comparison to NMR data. 2013, Pubmed
Jacobsen, The secret life of kinases: insights into non-catalytic signalling functions from pseudokinases. 2017, Pubmed
Jacobsen, HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death. 2016, Pubmed
Johnston, Necroptosis-blocking compound NBC1 targets heat shock protein 70 to inhibit MLKL polymerization and necroptosis. 2020, Pubmed
Kabsch, Integration, scaling, space-group assignment and post-refinement. 2010, Pubmed
Kitur, Necroptosis Promotes Staphylococcus aureus Clearance by Inhibiting Excessive Inflammatory Signaling. 2016, Pubmed
Kornev, Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism. 2006, Pubmed
Li, The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. 2012, Pubmed
Littlefield, Structural analysis of the EGFR/HER3 heterodimer reveals the molecular basis for activating HER3 mutations. 2014, Pubmed
Lupardus, Structure of the pseudokinase-kinase domains from protein kinase TYK2 reveals a mechanism for Janus kinase (JAK) autoinhibition. 2014, Pubmed
Madeira, The EMBL-EBI search and sequence analysis tools APIs in 2019. 2019, Pubmed
Manning, The protein kinase complement of the human genome. 2002, Pubmed
McCoy, Phaser crystallographic software. 2007, Pubmed
Mompeán, The Structure of the Necrosome RIPK1-RIPK3 Core, a Human Hetero-Amyloid Signaling Complex. 2018, Pubmed
Murphy, The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. 2013, Pubmed
Murphy, Live and let die: insights into pseudoenzyme mechanisms from structure. 2017, Pubmed
Murphy, A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties. 2014, Pubmed
Murphy, The Killer Pseudokinase Mixed Lineage Kinase Domain-Like Protein (MLKL). 2020, Pubmed
Murphy, Insights into the evolution of divergent nucleotide-binding mechanisms among pseudokinases revealed by crystal structures of human and mouse MLKL. 2014, Pubmed
Murphy, Bio-Zombie: the rise of pseudoenzymes in biology. 2017, Pubmed
Müller, Necroptosis and ferroptosis are alternative cell death pathways that operate in acute kidney failure. 2017, Pubmed
Nailwal, Necroptosis in anti-viral inflammation. 2019, Pubmed
Newton, Necroptosis and Inflammation. 2016, Pubmed
Pearson, EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation. 2017, Pubmed
Pearson, Down the rabbit hole: Is necroptosis truly an innate response to infection? 2017, Pubmed
Pefanis, Regulated necrosis in kidney ischemia-reperfusion injury. 2019, Pubmed
Petrie, Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies. 2020, Pubmed
Petrie, The Structural Basis of Necroptotic Cell Death Signaling. 2019, Pubmed
Petrie, Viral MLKL Homologs Subvert Necroptotic Cell Death by Sequestering Cellular RIPK3. 2019, Pubmed
Petrie, Conformational switching of the pseudokinase domain promotes human MLKL tetramerization and cell death by necroptosis. 2018, Pubmed
Pham, Viral M45 and necroptosis-associated proteins form heteromeric amyloid assemblies. 2019, Pubmed
Pierdomenico, Necroptosis is active in children with inflammatory bowel disease and contributes to heighten intestinal inflammation. 2014, Pubmed
Ribeiro, Emerging concepts in pseudoenzyme classification, evolution, and signaling. 2019, Pubmed
Rickard, RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis. 2014, Pubmed
Rickard, TNFR1-dependent cell death drives inflammation in Sharpin-deficient mice. 2014, Pubmed
Rodriguez, Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis. 2016, Pubmed
Samson, MLKL trafficking and accumulation at the plasma membrane control the kinetics and threshold for necroptosis. 2020, Pubmed
Sun, Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. 2012, Pubmed
Tanzer, Evolutionary divergence of the necroptosis effector MLKL. 2016, Pubmed
Tanzer, Necroptosis signalling is tuned by phosphorylation of MLKL residues outside the pseudokinase domain activation loop. 2015, Pubmed
Upton, Virus inhibition of RIP3-dependent necrosis. 2010, Pubmed
Vince, IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis. 2007, Pubmed
Wang, Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. 2014, Pubmed
Warnecke, PyTMs: a useful PyMOL plugin for modeling common post-translational modifications. 2014, Pubmed
Xie, Structural insights into RIP3-mediated necroptotic signaling. 2013, Pubmed , Xenbase
Zeqiraj, Structure of the LKB1-STRAD-MO25 complex reveals an allosteric mechanism of kinase activation. 2009, Pubmed
Zhao, Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis. 2012, Pubmed